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MB Sample ID: SA161886
Local Sample ID: | Mito_011_po |
Subject ID: | SU001794 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Genotype Strain: | HeLa cell line |
Gender: | Not applicable |
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Subject:
Subject ID: | SU001794 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Genotype Strain: | HeLa cell line |
Gender: | Not applicable |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
Mito_011_po | SA161886 | FL019126 | Mock(+) | Group |
Collection:
Collection ID: | CO001787 |
Collection Summary: | Mitochondria were isolated from HeLa cells using a mitochondria isolation kit for tissues (cat. no. 89874, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Mitochondrial pellets were washed and stored in 1xTBS buffer supplemented with 0.1% CHAPS on ice until further use. Isolated mitochondrial fractions were used for lipidomic analyses. |
Sample Type: | HeLa cells |
Treatment:
Treatment ID: | TR001807 |
Treatment Summary: | Conditional knockdown and reconstitution of PTPIP51 in HeLa cells were performed as previously reported (Bong et al, 2020). Huma PTPIP51-targeting small hairpin RNA (shRNA) sequences (5’-ATGACTTGATGCCACTATTTA-3’) were inserted into the Tet-pLKO-blasticidin vector. Lentiviruses were produced according to a method described previously (Kim et al, 2018). HeLa cells infected with lentiviruses were selected with blasticidin (10 μg/ml, Invitrogen, USA) for at least 7 days and named HeLa Tet-on-shPTPIP51 cells. The genes encoding human full-length PTPIP51 and PTPIP51_ΔFFAT were cloned into the pCAG-Flag-IRES-puro vector. HeLa Tet-on-shPTPIP51 cells were transfected with pCAG-Flag-IRES-puro empty vector (Mock), PTPIP51, and PTPIP51_ΔFFAT using Lipofectamine 3000 (Life Technologies, USA). Transfected cells were selected with puromycin (2 μg/ml, Amresco, USA) for at least 4 days. For endogenous PTPIP51 knockdown, doxycycline (1 μg/ml, Sigma-Aldrich, USA) was added every two days. Because the shRNAs were designed to target the 3’ UTR of PTPIP51, exogenously added constructs were not targeted. |
Sample Preparation:
Sampleprep ID: | SP001800 |
Sampleprep Summary: | For mitochondrial analysis, isolated mitochondria from 2x10^7 cells were mixed with 500 µl of 100 mM hydrochloric acid solution in methanol/water (8:2, v/v), 700 µl of chloroform and 500 µl of 100 mM hydrochloric acid solution in water. The mitochondrial sample was homogenized with 2.8 mm zirconium oxide beads for 5 min. After centrifugation at 30,130 xg and 4°C for 15 min, 600 µl of the lower phase was collected. Both the cell and mitochondrial extracts were dried under a gentle nitrogen stream at room temperature and reconstituted in 300 µL of isopropanol/acetonitrile/water (2:1:1, v/v/v). Eighty microliters of each sample was mixed with 20 µL of SPLASH LIPIDOMIX internal standard mix (Avanti Polar Lipids, USA). Finally, 5 µl of each sample was injected into the UPLC-QTOF MS system. |
Combined analysis:
Analysis ID | AN002797 | AN002798 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | Reversed phase | Reversed phase |
Chromatography system | Waters Acquity I-Class | Waters Acquity I-Class |
Column | Waters Acquity CSH C18 (100 x 2.1mm,1.7um) | Waters Acquity CSH C18 (100 x 2.1mm,1.7um) |
MS Type | ESI | ESI |
MS instrument type | QTOF | QTOF |
MS instrument name | ABI Sciex 5600 TripleTOF | ABI Sciex 5600 TripleTOF |
Ion Mode | POSITIVE | NEGATIVE |
Units | Peak area | Peak area |
Chromatography:
Chromatography ID: | CH002067 |
Chromatography Summary: | Chromatographic separation of lipids in cells and mitochondria was performed with an Acquity UPLC system (Waters, USA) using an Acquity UPLC CSH C18 column (2.1100 mm, 1.7 µm; Waters) at 55°C and a flow rate of 0.4 ml/min. The mobile phase for positive ion mode comprised 10 mM ammonium formate in water/acetonitrile (40:60, v/v) containing 0.1% formic acid (solvent A) and isopropanol/acetonitrile (90:10, v/v) containing 0.1% formic acid (solvent B). The mobile phase for negative ion mode comprised 10 mM ammonium acetate in water/acetonitrile (60:40, v/v) (solvent A) and isopropanol/acetonitrile (90:10, v/v) (solvent B). The UPLC gradient was programmed as follows: 40% to 43% B from 0 min to 2 min, 43% to 50% B from 2 min to 2.1 min, 50% to 54% B from 2.1 min to 12 min, 54% to 70% B from 12 min to 12.1 min, 70% to 99% B from 12.1 min to 18 min, 99% to 40% B from 18 min to 18.1 min, and 40% B for 2 min to equilibrate for the next run. |
Instrument Name: | Waters Acquity I-Class |
Column Name: | Waters Acquity CSH C18 (100 x 2.1mm,1.7um) |
Column Temperature: | 55 |
Flow Gradient: | 40% to 43% B from 0 min to 2 min, 43% to 50% B from 2 min to 2.1 min, 50% to 54% B from 2.1 min to 12 min, 54% to 70% B from 12 min to 12.1 min, 70% to 99% B from 12.1 min to 18 min, 99% to 40% B from 18 min to 18.1 min, and 40% B for 2 min to equilibrate for the next run. |
Flow Rate: | 0.4 ml/min |
Solvent A: | 40% water/60acetonitrile; 0.1% formic acid; 10 mM ammonium formate |
Solvent B: | 90% isopropanol/10% acetonitrile |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS002592 |
Analysis ID: | AN002797 |
Instrument Name: | ABI Sciex 5600 TripleTOF |
Instrument Type: | QTOF |
MS Type: | ESI |
MS Comments: | m/z range of 80-1500 The following parameter settings were used: ion spray voltage, 5500 V (positive mode) and 4500 V (negative mode); source temperature, 500°C; nebulizer gas pressure, 50 psi; drying gas pressure, 60 psi; and curtain gas pressure, 30 psi. An atmospheric pressure chemical ionization calibration solvent was used to maintain mass accuracy with an automated calibrant delivery system (Sciex). Information-dependent acquisition (IDA) was used to acquire MS/MS spectra for ions. All samples were pooled in equal amounts to generate quality control (QC) samples, which were injected after every 4 samples to calculate the coefficient of variation (CV) and assess analytical reproducibility. |
Ion Mode: | POSITIVE |
MS ID: | MS002593 |
Analysis ID: | AN002798 |
Instrument Name: | ABI Sciex 5600 TripleTOF |
Instrument Type: | QTOF |
MS Type: | ESI |
MS Comments: | m/z range of 80-1500 The following parameter settings were used: ion spray voltage, 5500 V (positive mode) and 4500 V (negative mode); source temperature, 500°C; nebulizer gas pressure, 50 psi; drying gas pressure, 60 psi; and curtain gas pressure, 30 psi. An atmospheric pressure chemical ionization calibration solvent was used to maintain mass accuracy with an automated calibrant delivery system (Sciex). Information-dependent acquisition (IDA) was used to acquire MS/MS spectra for ions. All samples were pooled in equal amounts to generate quality control (QC) samples, which were injected after every 4 samples to calculate the coefficient of variation (CV) and assess analytical reproducibility. |
Ion Mode: | NEGATIVE |